Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified kinase that phosphorylates and activates protein kinase B (PKB). Activation of PKB by insulin is linked to its translocation from the cytosol to the plasma membrane. However, no data are available yet concerning the localization of PDK1 in insulin-sensitive tissue. Using isolated adipocytes, we studied the effect of insulin and of an insulin-mimicking agent peroxovanadate on the subcellular localization of PDK1. In unstimulated adipocytes, overexpressed PDK1 was mostly cytosolic with a low amount associated to membranes. Peroxovanadate stimulation induced the redistribution of PDK1 to the membranes while insulin was without effect. This peroxovanadate effect was dependent on phosphatidylinositol 3,4,5 triphosphate [PtdIns(3,4,5)P3] production as inhibition of PtdIns 3-kinase by wortmannin or deletion of the PH domain of PDK1 prevented the peroxovanadate-induced translocation of PDK1. Further, peroxovanadate-treatment induced a tyrosine phosphorylation of PDK1 which was wortmannin insensitive and did not require the PH domain of PDK1. An inhibitor of Src kinase (PP2) decreased the peroxovanadate-induced PDK1 tyrosine phosphorylation and overexpression of v-Src stimulated this phosphorylation. Mutation of tyrosine 373 of PDK1 abolished the v-Src induced PDK1 tyrosine phosphorylation and partially reduced the effect of peroxovanadate. Our findings suggest that PDK1 could be a substrate for tyrosine kinases and identify Src kinase as one of the tyrosine kinases able to phosphorylate PDK1.